Introduction

Historic drawing comparing the size of the mature human oocyte with a spermatozoa

Prior to release from the ovary oocytes (eggs, ova) are arrested at an early stage of the first meiotic division as a primary oocyte (primordial follicle). Following puberty, during each menstrual cycle, pituitary gonadotrophin stimulates completion of meiosis 1 the day before ovulation. Early oocytes are also classified as immature (germinal vesicle (GV) or metaphase I (MI) stage). The breakdown of the germinal vesicle indicates a resumption of meiosis and the extrusion of the first polar body (1 PB) indicates completion of the first meiotic division in human oocytes.

The released oocyte is surrounded by a thick specialised extracellular matrix, the zona pellucida, that in turn is covered in layers of cells, the granulosa layer.

In an adult human female, the development of a primordial follicle containing an oocyte to a preovulatory follicle takes in excess of 120 days.

Some Recent Findings

Complete in vitro oogenesis: retrospects and prospects[1] "In reality the vast majority of oocytes formed from primordial germ cells (PGCs) will undergo apoptosis, or other forms of cell death. Removal occurs during germ cell cyst breakdown and the establishment of the primordial follicle (PF) pool, during the long dormancy at the PF stage, or through follicular atresia prior to reaching the ovulatory stage. A way to solve this limitation could be to produce large numbers of oocytes, in vitro, from stem cells. However, to recapitulate mammalian oogenesis and produce fertilizable oocytes in vitro is a complex process involving several different cell types, precise follicular cell-oocyte reciprocal interactions, a variety of nutrients and combinations of cytokines, and precise growth factors and hormones depending on the developmental stage. In 2016, two papers published by Morohaku et al. and Hikabe et al. reported in vitro procedures that appear to reproduce efficiently these conditions allowing for the production, completely in a dish, of a relatively large number of oocytes that are fertilizable and capable of giving rise to viable offspring in the mouse. The present article offers a critical overview of these results as well as other previous work performed mainly in mouse attempting to reproduce oogenesis completely in vitro and considers some perspectives for the potential to adapt the methods to produce functional human oocytes."

A stereological study on organelle distribution in human oocytes at prophase I[2] "The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distribution were observed between ooplasm regions: cortical vesicles (C: 1.3% versus SC: 0.1%, IC: 0.1%, P = 0.001) and medium-sized vesicles containing zona pellucida-like materials (C: 0.2% versus SC: 0.02%, IC: 0%, P = 0.004) were mostly observed at the oocyte cortex, whereas mitochondria (C: 3.6% versus SC: 6.0%, IC: 7.2%, P = 0.005) were preferentially located in the subcortex and inner cytoplasm, and SER very large vesicles (IC: 10.1% versus C: 0.9%, SC: 1.67%, P = 0.001) in the oocyte inner cytoplasm." Prophase I | Meiosis

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women[3] "Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo." (these cells described as oogonial stem cells (OSCs), are very rare—only about 1 out of 10,000 ovarian cells)

More recent papers

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Oogenesis

Secondary follicle with oocyte.

A human infant ovary histology, showing the large number of oocytes occupying the ovary cortical region. Compare this with a mature ovary and note the absence of any follicle development in the infant. These early oocytes remain at the diplotene stage of the meiosis I during development from fetal life and postnatal childhood, until puberty when the lutenizing hormone (LH) surges stimulate the resumption of meiosis.

The graph below shows the changes in human germ cell numbers in the ovary with age, peaking at about 7 million (occuring in early fetal development) and then decreasing by apopotic cell death. At puberty there remain only about 400,000 and only about 10% of these will be released through reproductive life. (More? Menstrual Cycle)

Meiosis

Human Oocyte metaphase of meiosis 2

In females, the total number of eggs ever to be produced are present in the newborn female initially arrested at the diplotene stage of the meiosis I from fetal life through childhood until puberty, when the lutenizing hormone (LH) surges stimulate the resumption of meiosis.

All eggs are arrested at an early stage (prophase I) of the first meiotic division as a primary oocyte (primordial follicle). Following purberty, during each menstrual cycle, pituitary gonadotrophin stimulates completion of meiosis 1 the day before ovulation.

In meiosis 1, a diploid cell becomes 2 haploid (23 chromosomes) daughter cells, each chromosome has two chromatids. One cell becomes the secondary oocyte the other cell forms the first polar body.

The secondary oocyte then commences meiosis 2 which arrests at metaphase and will not continue without fertilization.

At fertilization meiosis 2 completes, forming a second polar body. Note that the first polar body may also undergo this process forming a third polar body.

Polar Body

Human oocyte at metaphase II showing polar body at top

The breakdown of the germinal vesicle indicates a resumption of meiosis and the extrusion of the first polar body (1 PB) indicates completion of the first meiotic division in human oocytes. The polar body is a small cytoplasmic exclusion body formed to enclose the excess DNA formed during the oocyte (egg) meiosis and following sperm fertilization. There are 2-3 polar bodies derived from the oocyte present in the zygote, the number is dependent upon whether polar body 1 (the first polar body formed during meiosis 1) divides during meiosis 2. This exclusion body contains the excess DNA from the reductive division (the second and third polar bodies are formed from meiosis 2 at fertilization). These polar bodies do not contribute to the future genetic complement of the zygote, embryo or fetus.

Recent research in some species suggest that the space formed by the peripheral polar body (between the oocyte and the zona pellucia) can influence the site of spermatozoa fertilization.

The squared region of the cortical cap/ring is shown on the top, an actin cap (red) surrounded by a myosin II ring (green).

Assisted reproductive techniques involving intracytoplasmic sperm injection (ICSI) have looked at the "quality" of the polar body and found that the morphology is related to mature oocyte viability and has the potential to predict oocyte fertilization rates and pregnancy achievement.[7][8]

Calcium Release

Oocyte calcium ion (Ca2+) release occurs after spermatozoa fusion and is part of the reactivation of meiosis (arrested at metaphase II) and the primary block to polyspermy. Earlier in oocyte meiosis, between prophase I (germinal vesicle stage) and MII, this release mechanism is developed within the cell.

Oocyte cytoplasmic changes include:

endoplasmatic reticulum reorganization.

IP3 receptor increase in both number and sensitivity.

increase in calcium ion concentration.

Cortical Granules

Human oocyte (MII) showing cortical granules (green)

Mouse oocyte cortical granules (red)

The release of cortical granules by exocytosis, the "cortical reaction", occurs following spermatozoa fertilisation and is the main block to polyspermy by modifying the zone pellucida. These granules develop from the golgi apparatus initially forming smaller vesicles that coalesce to form mature membrane bound cortical granules (0.2 to 0.6 microns in diameter) located in the cortex of unfertilized oocytes. In mammals, cortical granule production in the developing follicular oocyte is an ongoing and continuous process, with newly synthesized granules translocating to the cortex until the time of ovulation.

Ovastacin

female mice lacking ovastacin do not cleave ZP2 after fertilisation[9]

Oocyte-Follicle Cell Interaction

The oocyte and the surrounding granulosa cells have a complex paracrine interactions during follicle growth and development. Oocyte maturation has been shown to depend on secretory products of both the granulosa and cumulus cells.

Oocyte Metabolism

In the mouse, the secondary follicle stage through to large antral follicle stage the oocyte has a highly oxidative metabolism. In contrast, the surrounding surrounding granulosa and cumulus cells are highly glycolytic. In this second group, the cumulus cells are found to be more glycolytic than the granulosa cells.[11]

oxidative metabolism - requires oxygen (aerobic) to make energy from carbohydrates (sugars) into pyruvate that passes into the mitochondria where it is fully oxidised by oxygen into carbon dioxide. Also called aerobic metabolism, aerobic respiration, and cell respiration.

glycolytic metabolism - requires no oxygen (anaerobic) to make energy from carbohydrates (sugars) into pyruvate that is reduced by adenine dinucleotide hydride (NADH).

In the cat oocyte, in pre-antral oocytes mitochondria have a homogeneous distribution throughout the cytoplasm. In the antral stage they have relocated to a mainly pericortical distribution.[12]

Historic Images

Historic Disclaimer - information about historic embryology pages

Pages where the terms "Historic Textbook" and "Historic Embryology" appear on this site, and sections within pages where this disclaimer appears, indicate that the content and scientific understanding are specific to the time of publication. This means that while some scientific descriptions are still accurate, the terminology and interpretation of the developmental mechanisms reflect the understanding at the time of original publication and those of the preceding periods, these terms and interpretations may not reflect our current scientific understanding. (More? Embryology History | Historic Embryology Papers)

Terms

antral follicle - (secondary) the stage following preantral in the decription of the sequence ovarian follicle development.

antrum - (L. a cave), cavity; a nearly-closed cavity or bulge. In the ovary this refers to the follicular fluid-filled space within the follicle.

atretic follicle - An ovarian follicle that fails to mature and degenerates. Also called "atresia" refering to the process of degeneration of the ovarian follicle. This process can occur at any stage of follicle development (folliculogenesis).

clomiphene citrate - drug taken orally to promote the process of follicle/egg maturation.

corona radiata - Layer of follicle cells of cumulus oophorus remaining attached to zona pellucida of oocyte after ovulation. Also called granulosa cells.

corpus luteum - (L. corpus = body, L. luteum = yellow) The remains of ovarian follicle after ovulation that acts as an endocrine organ supporting pregnancy and preventing menstruation (loss of the endometrial lining). de Graaf first observed it in the ovary of a cow as a yellow structure.

cortical - (L. corticalis) at the outside (like the bark of a tree), usually combined with medulla meaning the core.

follicle - (L. folliculus = little bag,dim. of L. follis). A structure which develops in the ovary and contains a developing egg (oocyte).

follicle stimulating hormone - (FSH, gonadotropin) A glycoprotein hormone secreted by anterior pituitary (adenohypophysis gonadotrophs, a subgroup of basophilic cells) and acts on gametogenesis and other systems in both males and females. Females, FSH acts on the ovary to stimulate follicle development. Males, acts on the testis Sertoli cells to increase androgen-binding protein (ABP) that binds androgens and has a role in spermatogenesis.

follicular fluid - the fluid found in the antrum of a secondary follicle. Secreted by cells in the wall of the follicle. This fluid is released along with the oocyte at ovulation.

germinal epithelium - cellular component covering surface of ovary, it is continuous with mesothelium covering mesovarium. Note that it is a historical misnomer, as it is not the actual site of germ cell formation.

Graafian follicle - named after Regnier de Graaf (1641-1673), an historic Dutch physician embryologist who studied pregnancy using rabbits.

granulosa cells - the supporting cells that surround the developing egg within the follicle thecal layers.

Izumo1 - a protein located on the equatorial segment of acrosome-reacted spermatozoa recognizes its receptor Juno, on the oocyte surface, for plasma membrane binding and fusion. Named for a Japanese shrine dedicated to marriage. OMIM609278

Juno - (folate receptor-δ; FOLR-δ) a glycophosphatidylinositol (GPI)-anchored, cysteine-rich glycoprotein on the oocyte surface for fertilisation that is the receptor of Izumo1 on the spermatozoa, for plasma membrane binding and fusion. OMIM615737

luteinizing hormone - (LH, gonadotropin, lutropin, Interstitial Cell Stimulating Hormone, ICSH) glycoprotein hormone releasd from anterior pituitary hormone that acts on the gonad and has a role in male and female reproduction. Female, LH triggers ovulation (release of the oocyte). Male, LH stimulates testis interstital cell (Leydig cell) production of testosterone. Have been used clinically in humans for the treatment of female infertility.

mesovarium - mesentry of the ovary formed from a fold of the broad ligament that attaches the ovary.

medullary - (L. medius = in the middle) relating to the medulla; pith, marrow, inner portion of an organ. Usually combined with cortex (cortical) meaning the outer layer.

oocyte - (Greek, oo = egg, ovum) The term used to describe the haploid egg or ovum formed within the ovary (female gonad) and released to enter the uterine tube and be transported to the uterus. The mature oocyte is the cell released from the ovary during ovulation.

oogonia - (Greek, oo = egg) diploid germ cells within the ovary (female gonad) which provide the primary oocytes for oocyte (egg) formation. In humans, all oogonia form primary oocytes within the ovary before birth.

oolemma - (zona pellucida, vitelline membrane).

oophorus - (Greek, oo = egg + phorus = carrying, egg-bearing) cumulus oophorus, used to describe the granulosa cells within the follicle that tether or link the oocyte to the wall of the follicle.

ovulation - release of the oocyte from the mature follicle. In humans generally a single oocyte is released from a cohort of several maturing follicles.

preantral follicle - (primary) the stage following primordial in the description of the sequence ovarian follicle development.

primary follicle - (preantral) the stage following primordial in the description of the sequence ovarian follicle development.

primordial follicle - the first stage in the description of the sequence ovarian follicle development. Present in the ovary from birth, located in the stroma of the ovary cortex beneath the tunica albuginea. The primordial follicle is the oocyte and the surrounding follicular cells.

primordial germ cell - oocyte present in the primordial follicle ovary from birth, located in the stroma of the ovary cortex beneath the tunica albuginea. The primordial follicle is the oocyte and the surrounding follicular cells.

secondary follicles - the stage following primary in the description of the sequence ovarian follicle development.

stromal cells - in the ovary, cells surrounding the developing follicle that form a connective tissue sheath (theca folliculi). This layer then differentiates into 2 layers (theca interna, theca externa). This region is richly vascularized and involved in hormone secretion.

superovulation therapy - a fertility drug treatement (oral clomiphene citrate and/or injectable FSH with or without LH) aimed at stimulating development/release of more than one follicle during a single menstrual cycle.

tertiary follicle - (preovulatory, Graffian) the stage following secondary in the description of the sequence ovarian follicle development.

theca folliculi - stromal cells in the ovary, cells surrounding the developing follicle that form a connective tissue sheath. This layer then differentiates into 2 layers (theca interna, theca externa). This region is vascularized and involved in hormone secretion.

theca interna - stromal cells forming the inner layer of the theca folliculi surrounding the developing follicle. This vascularized layer of cells respond to LH (leutenizing hormone) synthesizing and secreting androgens which are processed into estrogen.

transzonal projection - (TZP) ovarian follicle term describing the cellular membraneous extension from the granulosa cell through the zona pellucida to the oocyte cell membrane where it forms gap junctions or adherens junctions allowing signalling and adhesion between the two cells.

uterus - site of embryo implantation and development. Uterine wall has 3 major layers: endometrium, myometrium, and perimetrium. Endometrium can be further divided into the functional layer (shed/lost during menstruation) and basal layer (not lost during menstruation).